Protein research is the mainstay for understanding the machinery of biological systems. Recently, proteomics research and protein biomarker research have made a strong impact in furthering our knowledge of biological systems, and holds great promise for research into diseases, hopefully leading to useful drug treatments and diagnostics.
Traditionally, electrophoresis has been a key laboratory technique for separating and studying proteins, and has been used as an analytical technique itself. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was one of the earliest electrophoretic methods for separating complex protein mixtures according to their molecular weight. Isoelectric focusing is another electrophoretic technique that separates mixtures according to their charge. A very powerful separation technique is 2-dimensional (2D) electrophoresis, in which the mixture is first fractionated by isoelectric focusing in a gel strip, and then the strip is equilibrated with SDS, mated with a polyacrylamide slab gel, and the charge separated mixture is further separated according to molecular weight, creating a 2-dimensional image of the mixture with charge separation in the x-direction and molecular weight separation in the y-direction. The result of a 2D procedure is a spot image, and further analysis needs to be done on these spots for actual protein identification. Two approaches are antibody probing methods, like western blotting, or amino acid sequencing.
Recently, mass spectrometry (MS) has become a very important tool for identifying proteins by sequence. Total mass, and fragment masses are used to deduce the amino acid sequence, and hence protein identity. Most recently, as the capabilities of the MS instrumentation have improved, the MS has increasingly been taking the place of the second dimension of 2-D electrophoresis. Tandem liquid chromatography-mass spectrometry (LC-MS) is the workhorse of protein identification today. The LC fractionates samples by hydrophobicity, and the MS identifies the proteins associated with the fractions. Samples analyzed by LC-MS can be more complex as instrumentation improves, analyzing up to hundreds of proteins per sample. However, typical biological samples contain thousands of proteins. Thus, some fractionation prior to LC-MS analysis is still required in the sample preparation workflow. Charge-based separations are considered to be important, because of the additional structural information that can be deduced from charge.
The methods used to prepare complex protein or peptide samples for subsequent immunoassay or LC-MS analyses were generally developed as analytical tools themselves. Consequently, they have high separation resolution, tend to be complex to run, and are usually expensive on a per sample basis. With advancements in, for example LC-MS, only crude fractionation is necessary in the preparative steps, and with the proliferation of high throughput workflows there is a need for effective, rapid, low-cost, charge-based fractionation. Also, it is advantageous to have the analytes separated in a gel-free system to minimize losses, and to simplify sample clean-up prior to any LC-MS procedure.
Strong cation exchange chromatography is a charge-based protein sample fractionation technique. This has the advantage that chromatography equipment is prevalent in the laboratory environment, and it is easily automated. It suffers the disadvantage that many of the analyte species irreversibly bind to the column, which limits the quantitative capabilities of the method. Isoelectric focusing is the other charge-based separation process frequently used in the biochemistry laboratory.
Electrophoretic separations dominate charge-based separation methods. Isoelectric focusing is done in a pH gradient. Charged molecules migrate in an electric field within the gradient where the overall charge of an amphoteric molecule is affected by pH. An amphoteric molecule gains positive charges at pH values below the pKas of the charged moieties within the molecule, hence migrates toward the cathode, or attains negative charges at higher pH values, hence migrates toward the anode. At some intermediate pH, positive and negative charges balance and there is no net movement in the electric field, the isoelectric point (pI). In isoelectric separation, a complex mixture is separated into bands along a pH gradient corresponding to each component's isoelectric point. The pH gradients are established in two basic ways: (1) with carrier ampholytes, a complex mixture of amphoteric molecules line up in the electric field to give a piecewise discrete pH gradient; and (2) immobilized pH gradients fabricated into a polyacrylamide gel by incorporating acrylamido buffers, monomers with acids or bases pendant on the side chains, which locally buffer a specific pH. Frequently, a combined approach is used.
The most typical configuration for isoelectric focusing is the IPG strip (immobilized pH gradient). This format requires relatively large sample volumes (10-50 μg), long running times (5 to 24 hours), and high applied voltage (1,000-10,000 Volts). This method has exquisitely fine separation resolution to about 0.01 pH units, and has been used as an analytical tool in protein research. The separated mixture is in a gel matrix, however, and needs to be recovered and cleaned prior to any subsequent analysis, such as LC-MS. This is usually accomplished by running an SDS-PAGE second dimension. Protein spots in the 2D-SDS-PAGE need to be excised, cleaned of SDS and eluted prior to LC-MS analysis. Non-specific adsorption of protein to the polyacrylamide matrix, and the workflow complexity of recovering the separated proteins to a liquid phase make a gel-free separation technique desirable. Additionally, the high applied voltage poses a safety hazard, and the power supplied to these systems causes them to heat, which may damage proteins or peptides, and requires active cooling.
Capillary isoelectric focusing (CIEF) is a newer technology conducting the separation in a capillary using carrier ampholytes to create the pH gradient, usually in a gel-free medium. CIEF utilizes very small sample sizes on the order of 1 μL. Proteins or peptides may be recovered by pushing the separated mixture out of the capillary along the direction of the separation. This is not easily accomplished, because the capillary ends are in contact with the anode and cathode buffer pools. So, the capillary must be disconnected from the electrophoresis running system and attached to a mechanical pumping system to recover the contents of the capillary. Usually, however, the analytes are visualized in place using fluorescent tags.
In WO 2008/006201 a CIEF device is taught wherein the analytes can be removed from the capillary via a single cross-flow channel. This geometry requires a complex fluid handling system to hydraulically position a segment of the separated analyte over the cross-flow extraction zone prior to the application of an extraction pressure to recover only that separated segment. The flow characteristics of such a device are designed so that it is impossible to simultaneously and uniformly recover all of the separated segments.
U.S. Pat. No. 7,655,477 B1 teaches a CIEF system with a multiplicity of side channels that recover separated analytes. This device requires a series of buffers of different ionic strengths to coordinate flow through the device, and the device does not intend to recover the samples for subsequent downstream analysis. Instead, UV-Visible spectra are obtained for the separated analytes within the side channels. It would be difficult to physically recover analytes because all of the movement is accomplished by electrophoretic or electroosmotic movement, and the analytes would be lost to the electrode buffer or oxidized at the electrode itself.
Free-flow electrophoresis (U.S. Pat. Nos. 5,275,706 and 6,328,868) is another gel-free separation method intended for the purification of large samples. Being gel-free is an advantage for certain protein or peptide workflows prior to subsequent immunoassay or mass spectrometry analysis. Free-flow electrophoresis (FFE) has not been well adopted because the equipment is cumbersome to use and expensive to purchase. As a continuous flow method, the equipment is difficult to set up, balance and calibrate. Furthermore, it requires a large sample volume (0.1-10 mg usually at concentrations on the order of 1 mg/mL). The method greatly shortens run times to 0.5-1 hours, and requires about 2,000 Volts to accomplish the separation at a resolution of about 0.1 pH units.
Micro FFE (μFFE) devices have been reported (e.g., S. Köhler, C. Weilbeer, S. Howitz, H. Becker, V. Beuhausen and D. Belder, PDMS free-flow electrophoresis chips with integrated partitioning bars for bubble segregation, in Lab on a Chip, 2011, 11:181). Although these address the FFE drawbacks of sample size and high voltage (about 100 V), they retain the fluidic complexity of FFE, requiring the flow rates of multiple inlet and outlet ports to be balanced.
There are numerous refinements of the FFE concept. See for example, U.S. Patent Application 2010/0252435 A1 and U.S. Pat. No. 6,328,868. These refine the FFE concept with additional flows to balance the system to deal with electroosmotic flow, operational variations, or with buffer systems that refine the pH gradient, but do not address the operational complexity or equipment expense of running FFE.
Multi-compartment electrolyzers (MCE) are large-scale isoelectric separation devices. These are comprised of liquid compartments separated by permeable membranes that allow the analyte molecules to migrate from compartment to compartment. The pH in each compartment is controlled by charged membranes (for example U.S. Pat. No. 4,971,670), and/or by various components in the buffer systems (for example U.S. Pat. Nos. 4,362,612 and 6,638,408). The initial intent was for purifying large quantities of peptides or proteins for non-MS uses. Consequently, these systems tend to be too large for analytical sample preparation. The MCE format is useful for fractionating complex samples prior to LC-MS because the fractionated material can be recovered in a gel-free, liquid phase. MCEs suffer the drawback of needing large samples (many milligrams), and generally need many hours to run, and have only crude pH resolution of about 0.1-1.0 pH units. An additional disadvantage is that proteins and peptides frequently adhere to the membranes and are lost.
Yet another approach is the Offgel™ system (Proteomics 2002, 2, 151-156 and Electrophoresis 2003, 24, 3-11) manufactured by Agilent Technologies (Santa Clara, Calif.). This is essentially an MCE wherein a linear array of open-bottomed chambers is placed on top of an IPG strip. The pH of each chamber is controlled by the average pH of the IPG segment over which it lies, and the membrane between each chamber is the IPG segment beneath the wall between two adjacent chambers. This is a very effective system for fractionating complex samples to a pI resolution of about 0.3 pH units. The fractions are recovered in liquid for relatively direct incorporation into an LC-MS workflow. The Offgel system still requires large samples, less than the traditional MCE, but more than a typical IPG strip. Long separation times (12-24 hours) and high voltages (up to 10,000 Volts) are additional disadvantages.
Ampholyte-free separations are advantageous for certain downstream analytical methods, such as mass spectrometry, since ampholytes interfere with protein and peptide mass spectra. U.S. Pat. Nos. 5,447,612 and 7,615,354 B2 and U.S. Pat. Appl. 2010/0252435 A1 all suggest similar buffer systems based on mixtures of organic acids and bases.
There is a need in the protein and peptide workflows, such as LC-MS, for a technology that can fractionate a complex sample in a short time, on the order of 1 hour; can provide charge information; can use a small sample, less than about 10 μg; does not require high voltages, preferably less than 200 Volts; has a high reproducibility; and is easy to use.